An immune signal from immunoglobulin G (hereinafter, referred to as IgG) is transmitted by binding of IgG after antigen trapping to an Fc receptor on the surface of an immunocyte. The Fc receptor is a group of protein molecules which binds to the Fc region of IgG, and each molecular species has an Fc recognition domain belonging to an immunoglobulin super family and recognizes a single variety of immunoglobulin or the immunoglobulin belonging to the same subtype. This determines which accessory cell is recruited in each immune response (Non-Patent Literature 1). The Fc receptor can be further classified into subtypes and it has been reported that FcγRI, FcγRIIa, FcγRIIb, and FcγRIII exist as receptors for IgG (Non-Patent Literature 1). Especially, the binding affinity of FcγRI to IgG is high and the equilibrium dissociation constant (KD) thereof is 10−8 M or less (Non-Patent Literature 2).
FcγRI is divided roughly into a signal peptide region, an extracellular region, a transmembrane region, and an intracytoplasmic region. The binding thereof to IgG occurs between the Fc region of IgG and the extracellular region of FcγRI, and subsequently the signal of their binding is transmitted into cytoplasm. FcγRI includes two kinds of subunits, an α chain directly associated with binding to IgG, and a γ chain. The γ chain forms a homodimer with a covalent bond through cysteine on the boundary between the transmembrane region and the extracellular region (Non-Patent Literature 1). The amino acid sequence and the gene base sequence of the α chain of FcγRI have been revealed by Non-Patent Literature 3, and thereafter an example of the expression thereof has been reported by genetic modification techniques with E. coli (Patent Literature 1) or animal cells as host cells (Non-Patent Literature 4).
The protein which constitutes the extracellular region of FcγRI (hereinafter, referred to as an Fc binding protein) as described above has excellent ability to identify a human antibody on the basis of high affinity. Based on this high affinity, the method for utilizing the Fc binding protein as a ligand of affinity chromatography used in a process of manufacturing diagnostic reagents, tools for research of antibody drugs, or antibody drugs such as of IgG, has been reported (Patent Literature 1).
The Fc binding protein is a protein originated from a protein which works in a human living body, and it has stronger tendency to be denatured by heat and extreme change of pH, etc. than a protein present outside a living body such as on the outer surface of a bacterial cell. On the other hand, when the Fc binding protein is used as a ligand for affinity chromatography to manufacture IgG, the Fc binding protein is required to have stability to acid since the ligand may be exposed to a solution of low pH, such as a citric acid buffer solution, in eluting adsorbed IgG through chromatography procedure using a gel with the ligand immobilized thereto. In addition, the Fc binding protein is also required to have stability to alkali since the ligand may also be exposed to a solution of high pH, such as a sodium hydroxide solution, when the gel is washed or regenerated. Further, in view of the long-term storage of the gel, the Fc binding protein is also required to have stability to heat.
To industrially utilize a biological substance such as a protein, it may be required that the native structure of the biological substance is modified to newly produce a substance which is stable under the predetermined conditions. Regarding an enzyme protein, etc., many examples have been reported in which a mutation is artificially introduced into a polynucleotide encoding the enzyme protein and a mutant which acquires a desired trait is obtained after screening. However, as for the Fc binding protein which is a receptor protein, no example of modification has been reported in which stability to heat, acid, or alkali is increased and thus no example has been industrially utilized so far.